Project Summary/Abstract The alpha herpesviruses, which includes important human pathogen herpes simplex virus 1 (HSV-1), are among the very few viruses that have evolved neuron-specific viral mechanisms to exploit highly-specialized neuronal cell biology. During the natural course of disease, alpha herpesviruses infect sensory or autonomic neurons, and establish a life-long latent infection in the peripheral nervous system. Upon reactivation, the virus can return to peripheral tissues, causing herpetic or zosteriform lesions, or it can spread to the central nervous system, causing severe herpes encephalitis. This proposal focuses on three major steps in the HSV-1 replication cycle, where virus particles must interact with specialized neuronal systems: 1. long-distance post- entry axonal transport; 2. long-distance post-replication transport of progeny particles towards axonal sites of egress; 3. polarized viral egress via exocytosis from axons, dendrites, or cell bodies. In Aim 1, we will identify sites of HSV-1 particle exocytosis in neurons, and determine the molecular/cellular mechanisms of HSV-1 egress from axons, dendrites, or cell bodies. In Aim 2, we will investigate the structural biology of HSV-1 axonal transport by cryo electron microscopy (cryoEM). My prior education and research experience has focused on studying viruses from a molecular and cell biological perspective, in particular, studying the interactions of enveloped viruses with host membrane systems. In the course of my graduate and postdoctoral research, I have developed expertise in using specialized microscopy methods to spatiotemporally dissect particular steps in the virus replication cycle. In the current postdoctoral phase of my career, I have developed a novel live-cell fluorescence microscopy assay of virus egress based on a specialized fluorescence microscopy method and a novel pH-sensitive fluorescent probe. In addition, I am currently developing new cryoEM tomography methods to investigate axonal transport of virus particles. This proposal seeks to extend these methods to accomplish the proposed aims. My scientific background makes me well suited to carry out the proposed research. This K22 Career Transition Award will help me to achieve my scientific and career goals, which include transitioning to an independent position as an assistant professor, and establishing an independent NIH-funded research program focused on neurovirology.